Studies of possible preventive effects of prenatal retinoic acid treatment on genetic birth defects in mice.
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Animals↗
Biomedical subjects
D M Juriloff
Publications and source records attributed to D M Juriloff.
Retinoic acid, cortisone, or thyroxine suppresses the mutant phenotype of the eyelid development mutation, lgMl, in mice.
The development of the mammalian eyelid is one of the last major morphogenetic events during prenatal development. The eyelids originate as outgrowths of epithelium and mesenchyme above and below the eye; they grow and flatten across the eyes, and meet and fuse firmly but temporarily until several days after birth in mice. In mouse lidgap mutants, eyelid development fails. Previously, we have shown that normal eyelid development can be induced in lidgap mutants by maternal treatment with cortisone or thyroxine. These hormones have been reported to interact with members of a receptor superfamily, and their response elements have been shown to induce transcription of hormone-regulated genes; retinoic acid also interacts with members of this superfamily. The purposes of our study were to examine the joint effect of thyroxine and cortisone on induced eyelid development in lidgap-Miller mutant fetuses (lgMl/lgMl), to test for synergism, and also to test whether retinoic acid also induces eyelid development in this mutant. We found that the combined effect of thyroxine and cortisone was significantly greater than that of either hormone alone, but that the dose-response slopes (log dose, probit scale) for eyelid development induced by cortisone alone or by cortisone preceded by 0.1 mg thyroxine were parallel, suggesting that their action is additive, not synergistic. We found that retinoic acid administered on day 14 of gestation induced eyelid development in lgMl/lgMl mutant fetuses, with a significant dose response and ED50 of 24 +/- 6 mg/kg. The response was treatment-time dependent, with optimal responses after day 14 or 11 treatment.(ABSTRACT TRUNCATED AT 250 WORDS)
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Ataxia and a cerebellar defect in the exencephaly-prone SELH/Bc mouse stock.
SELH/Bc inbred mice have an abnormal mechanism of anterior neural tube closure and 10-20% of embryos have a lethal neural tube closure defect, exencephaly. Our previous studies have focused on this multifactorial threshold trait. However, SELH mice are also characterized by another trait that also shows non-Mendelian transmission ratios, an ataxia recognized in juvenile and adult mice. Here we report our first genetic and morphological studies of the ataxia trait. Recent pedigree records for the SELH colony showed that 7% of the 467 weaned progeny from normal breeding pairs were ataxic; 17 of the 20 pairs produced ataxic progeny. This result was statistically consistent with the hypothesis that all SELH mice have the ataxic genotype, which is expressed in only 7% of them. Genetic studies of an outcross to a normal strain and the subsequent F2 and testcross of the F2 were also done. The results were consistent with a one or two gene locus cause of liability to ataxia in SELH mice. The genetic correlation between exencephaly production and ataxia production for a sample of nine F2 males was 0.35, as expected if both traits are caused by the same genes, but was not statistically significant. In another approach, we examined the morphology of brains from normal and ataxic adult SELH mice. All 20 brains from non-ataxic SELH mice were morphologically normal. In all 18 brains from ataxic SELH mice the cerebellum was abnormal, lacking the vermis, and characterized by a midline fissure. This phenotype in mice has previously been known in Mendelian mutants at the Wnt-1 locus.(ABSTRACT TRUNCATED AT 250 WORDS)
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Histological study of the cranial neural folds of mice genetically liable to exencephaly.
The SELH/Bc (SELH) inbred stock of mice has a high liability to the neural tube closure defect, exencephaly. All SELH embryos close their cranial neural tubes by an abnormal mechanism, lacking elevation and initiation of fusion in the posterior prosencephalon/anterior mesencephalon region. Most embryos complete closure of the cranial neural tube by extension of a more rostral site of fusion, but in 10-20% this process fails, and the embryos are subsequently exencephalic. In this study, transverse histological sections of the cranial neural folds of SELH embryos at the 3-5, 6-8, and 9-11 somite stages were compared to those of two strains with normal neural tube closure, ICR/Bc and LM/Bc. At all stages, consistent morphological differences were observed between SELH and the two normal strains. In 3-5 somite SELH embryos, the divergence of the folds from the neural groove is more angular, the folds are flatter, and their lateral tips appear "hooked" downward. In 6-8 somite SELH embryos, the lateral tips of the folds appear more elongated and in the prosencephalon they are less elevated than in the normal strains. The boundary between neuroepithelium and mesenchyme or surface ectoderm tends to be less clear than normal in SELH lateral tips. In 9-11 somite SELH embryos, divergence of the folds from the neural groove continues to be angular and the lateral folds are splayed horizontally. In addition, the lateral surface ectoderm is abnormally indented and the neuroepithelium/surface ectoderm boundary is more ventral and lateral in SELH than in ICR/Bc and LM/Bc. The hypothesis that the defect in SELH cranial neural folds might involve the cytoskeleton was tested using a fluorescent probe for filamentous actin in 7 somite SELH and ICR/Bc embryos. The actin staining pattern in SELH embryos was like that of normal ICR/Bc embryos, with a strongly staining apical concentration in the neuroepithelium. This suggests that there is no gross cytological abnormality within the neuroepithelium, but does not rule out more subtle defects, such as those involving cytoskeletal function.
Animals↗
Further genetic studies of the cause of exencephaly in SELH mice.
We have developed an inbred stock of mice called SELH that has a high frequency of the neural tube defect exencephaly at birth. A previous genetic study indicated that the exencephaly is due to two to three additive loci differing between SELH and a closely related normal strain, ICR/Bc, but this analysis was not designed to detect genetic maternal effects. Recently, we demonstrated that there is genetic polymorphism among normal mouse strains leading to differences in site of initiation of closure of the cranial neural tube. In the present study, an inbred substrain of SELH mice, with 24% exencephaly among embryos, was crossed with an unrelated normal strain, SWV/Bc, and the frequency of exencephaly in subsequent generations used to extend our understanding of the genetic cause of exencephaly in SELH mice. The purposes of the genetic studies reported here were twofold. First, based on the influence of genetic maternal effects on other genetically complex birth defects in mice, we hypothesized that the exencephaly of SELH mice would exhibit strong genetic maternal effects. This hypothesis was tested by comparisons among the four possible reciprocal backcrosses to SELH. The result was an overall frequency of 2.3% exencephaly in first backcross embryos with no difference among the four crosses and no evidence of genetic maternal effects. Second, the frequency of exencephaly recovered in the backcross and F1 embryos was compared with the previous genetic study and with various genetic models. The frequencies were similar to those obtained from the cross to ICR/Bc mice and were compatible with a hypothesis of additive gene action at a few loci.(ABSTRACT TRUNCATED AT 250 WORDS)
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Studies of a spontaneous lethal mutation at the albino locus in SELH/Bc mice.
We report a new mutation at the albino locus in SELH/Bc mice. The mutation arose spontaneously in a male mouse that appeared to be a somatic and germ line mosaic for a new albino (c) allele, provisionally named cBc. The mutation is a recessive lethal, causing embryonic death soon after implantation. We have shown that there is no detectable activity of the Mod-2 allele in cis with the mutation and conclude that the mutation is probably a deletion that includes the c locus, the Mod-2 locus, the intervening 2 cM, and at least one locus essential for postimplantation embryonic survival, either proximal to the c locus or distal to the Mod-2 locus. This new mutation is similar to most previously reported spontaneous mutations at the albino locus in that it arose in a somatic and germ line mosaic mutant animal but differs from them in that it is an embryonic lethal when homozygous and is apparently a deletion. SELH/Bc mice appear to have a high mutation rate. This lethal albino mutation that appears to be a postmeiotic deletion should be useful in the search for the mechanism of mutagenesis in SELH/Bc mice. It may also be useful in mapping essential genes in the c-locus region.
Albinism↗
Studies of the effect of retinoic acid on anterior neural tube closure in mice genetically liable to exencephaly.
Previously we have shown that all SELH/Bc mouse embryos close their anterior neural tubes by an abnormal mechanism and that 10-20% of SELH/Bc embryos are exencephalic. The purposes of these studies were (1) to observe the effects of retinoic acid on the frequency of exencephaly in SELH/Bc embryos; (2) to compare the SELH/Bc response with those of normal strains and of other neural tube mutants; and (3) to compare, between SELH/Bc and a normal strain (SWV/Bc), the effects of retinoic acid on morphology of the closing anterior neural tube. SELH/Bc was more liable to retinoic acid-induced exencephaly than were normal strains. After maternal treatment with 5 mg/kg retinoic acid on day 8.5 of gestation, 53% of SELH/Bc embryos had exencephaly, compared with 22% in ICR/Bc and 14% in SWV/Bc. When these results were transformed according to the assumptions of the developmental threshold model, the effects of genotype and retinoic acid appeared to be additive. Similar treatment on day 9 or 10 of gestation had little or no effect on the frequency of exencephaly in SELH/Bc mice. These results are similar to the reported responses of the curly-tail and Splotch mutants, where frequencies of spina bifida but not exencephaly were decreased. This pattern suggests that studies of effects of periconceptional vitamin treatment on risk of human neural tube defects should consider anencephaly and spina bifida separately. The study comparing the morphology of anterior neural tube closure in SELH/Bc and normal SWV/Bc embryos showed that retinoic acid delays the elevation of the mesencephalic neural folds. This results in a "stalling" of many embryos in the first steps of neural tube closure, with their neural folds remaining convex and splayed wide apart. The delay in fold elevation was superimposed on the different closure patterns of the two strains. The overall conclusion is that there is no nonadditive interaction in the parameters studied between retinoic acid treatment and the SELH/Bc genotype.
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Normal mouse strains differ in the site of initiation of closure of the cranial neural tube.
The scanning electron microscopic study of day 9 embryos reported here documents differences among normal mouse strains in morphology of cranial neural tube closure. The site of initiation of contact and fusion of the cranial neural folds, previously defined as Closure 2 (Macdonald et al., '89), is located in the region of the junction between the forebrain (prosencephalon) and midbrain (mesencephalon) in three normal strains: LM/Bc, AEJ/RkBc, and ICR/Bc. However in a fourth normal strain, SWV/Bc, Closure 2 is initiated much further rostral, in the prosencephalic region. In addition, the anterior neuropore, rostral to Closure 2, closes late in ICR/Bc embryos, relative to the posterior progress of development of the Closure 2 seam. Initiation of closure from the most rostral end of the neural tube (Closure 3) appears to be relatively delayed in ICR/Bc embryos. We hypothesize that the observed genetic polymorphism in location of the first site of fusion between the cranial neural folds in normal mouse embryos may be one basis for differences among normal strains in liability to exencephaly induced by teratogens.
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Disruption of pattern formation in palatal rugae in fetal mice heterozygous for First arch (Far).
The purpose of this study was to document the extent of disruption in the pattern of palatal rugae caused by the presence of one copy of the First arch mutation. The palatal ruga pattern was found to be disrupted in 86% of 15- to 17-day mouse fetuses that were heterozygous for the First arch mutation in the ICR/Bc strain, compared with 9% in ICR/Bc fetuses of normal (+/+) genotype. This new observation in First arch heterozygotes, together with the previously reported dominant effects of the First arch mutation, particularly the bifurcation of the maxillary nerve (100% in both BALB/cGaBc and ICR/Bc strains), the disruption of maxillary vibrissa pattern (80% in ICR/Bc), and the hemifacial deficiency (38% in ICR/Bc), has led us to redefine the First arch mutation as a semidominant, Far. Like the other defects caused by Far, the rugal defects are in tissue derived from the embryonic maxillary prominence. The rugal defects observed in +/Far palates were always asymmetrical and most often involved fragmentation and misalignment of two or more of rugae 4-7. The relatively large degree of variation in ruga pattern observed in fetuses of normal genotype suggests that it is a less well canalized trait than the normal pattern of maxillary vibrissae which varies only in a few very specific and minor ways. The First arch mutation, which in heterozygotes disrupts pattern formation in both palatal rugae and maxillary vibrissae, can be used to study genetic control of pattern formation in mammalian embryos.
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Developmental study of neural tube closure in a mouse stock with a high incidence of exencephaly.
About 17% of embryos and fetuses in the SELH/Bc mouse stock have the anterior neural tube defect, exencephaly. No other malformations are seen. The genetic liability to exencephaly was shown to be probably genetically fixed in the SELH/Bc stock. This means that SELH/Bc embryos with successful neural tube closure are genetically the same as exencephalics. Females were significantly more likely to be affected than males (66% females). The pattern of morphological developmental events during anterior neural tube closure on days 8 and 9 of gestation was compared among 322 ICR/Bc (normal), 304 SWV/Bc (normal), and 265 SELH/Bc embryos. Anterior neural tube closure was found to follow a strikingly different pattern in almost all SELH/Bc embryos than in either of the normal strains or in previous published studies. SELH/Bc embryos lack the initial contact between the anterior folds in the posterior prosencephalon/anterior mesencephalon region (Closure 2). In spite of this, all but 17% manage to close the anterior neural tube by extending caudally the later occurring normal anterior zone of contact and fusion at the most rostral aspect of the prosencephalon (Closure 3) through the region of Closure 2 to meet the zone of closure of the rhombencephalon, Closure 4. Anterior neural tube closure was completed late, and in some SELH/Bc embryos, elevation and fusion in the mesencephalon did not occur at all. In histological sections of six- and eight-somite embryos, elevated numbers of pyknotic cells in the neuroepithelium and mesenchyme, and elevated numbers of unstained inclusions in the neuroepithelium were found; but their relationship, if any, to the abnormal pattern of neural tube closure is not clear.
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A scanning electron microscope study of fetal eyelid closure accelerated by cortisone in SWV/Bc mice.
Eyelid closure occurs earlier in SWV/Bc and CBA/J mouse fetuses whose mothers were treated with cortisone on day 14 of gestation than it does in fetuses from untreated mothers. Similar treatment prevents the open-eyes defect of lidgap-Miller mutant mice, but examination by scanning electron microscope (SEM) has shown differences in the periderm of the closing eyelids of the cortisone-treated mutant compared with those of untreated genetically normal fetuses. (Untreated mutant eyelids remain wide open and very abnormal). The present study has examined at the SEM level the accelerated eyelid closure of cortisone-treated normal strain, SWV/Bc, fetuses to investigate whether the differences from normal in the eyelids of treated lidgap-Miller fetuses are part of the mechanism of the cortisone "cure." At the SEM level, cortisone-accelerated eyelid closure of SWV/Bc fetuses is indistinguishable from that in untreated fetuses. This suggests that the early eyelid closure induced by cortisone in normal strain fetuses represents acceleration of the normal coordinated sequence of events that leads to closure, rather than an abnormality that fortuitously leads to closure. The data also indicate that the cellular abnormalities seen previously in treated lidgap-Miller mutant fetuses are a combination of 1) abnormalities due to the mutation that are not completely reversed by cortisone and 2) normal developmental stages that have become concurrent with the cortisone-induced late closure in lidgap-Miller mutant fetuses.
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Genetic analysis of the cause of exencephaly in the SELH/Bc mouse stock.
A new mouse stock, SELH/Bc, having a high liability to exencephaly has been developed. About 17% of SELH fetuses are exencephalic. The genetic cause of this exencephaly was investigated in a cross to a normal related ICR/Bc strain and in subsequent classical genetic crosses (F2, first and second backcrosses). The data were compared with a number of genetic models, including that of a single recessive mutation with 17% penetrance. The data did not fit single-locus inheritance. The expectations from the multifactorial threshold model based on an underlying quantitative liability trait with additive inheritance were found to fit the data very well. The number of loci involved was estimated to be about two or three. About 70% of exencephalic SELH fetuses are female, and there is no overall deficiency of males. The relatively higher risk in females was constant across the genetic backgrounds in the experiment. In summary, the liability to exencephaly in SELH mice appears to be a multifactorial threshold trait, and it therefore resembles human neural tube defects in type of genetic etiology. SELH therefore may be a valuable animal model in the study of neural tube defects.
Animals↗
Cleft palate: more genetic lessons from mice.
Cleft palate occurred in high frequency (14%) in the F2 generation of the cross between two stocks of mice, LGG and SELH, neither of which produces more than 2% cleft palate. The cleft palate trait results from a new combination of alleles that is not present in either parental stock. The lack of cleft palate in the F2 generation after outcrosses of both parental stocks to other strains shows that this new combination of alleles has specific contributions from both parental strains, and also that there must be at least two loci involved. A deficiency of Mod-1 homozygotes in the SELH/LGG F2 adults suggests that one of the loci involved may be linked to Mod-1 and that the number of loci involved is few. Significantly more F2 males (19%) than females (9%) were affected with cleft palate. The data can be explained by a two-locus epistatic model with a dominant mutation (P) at one locus that causes cleft palate when not suppressed by or compensated for by a dominant allele (S) at a second locus. The parental stocks would be PPSS and ppss. In the F2 generation, the new combinations PPss and Ppss would express cleft palate, a total expected of 19%. Similar new combinations of alleles at two loci may explain some instances of high occurrence of cleft palate or other developmental threshold traits in previously unaffected human families.
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A small region of chromosome 9 in liability to cleft palate (CP) in mice.
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Animals↗
Maternal treatment with cortisone accelerates eyelid closure and other developmental fusion processes in fetal mice.
Cortisone acetate administered to pregnant CBA/J and SWV/Bc mice at 30 to 100 mg kg-1 on day 14 of gestation causes accelerated development of eyelid closure, digit fusion and fusion of pinnae to the scalp on day 16 of gestation. Eyelid closure seems to be accelerated more than hindlimb digit fusion. The results support the hypothesis that the prevention of the open-eye birth defect in lgMl/lgMl mutant mice by cortisone is through a shift of eye closure to an earlier chronological or morphological stage, and that the lgMl mutation causes delay in eyelid development and closure. Most previous studies of the effects of glucocorticoids on morphological development have focussed on high doses that induce defects, such as cleft palate, and on treatment earlier in gestation. In the studies reported here, lower doses were used and an acceleration of apparently normal development was observed. The results support the possibility that the gene regulatory effects of physiological levels of glucocorticoids are involved in normal morphological development of mammalian fetuses. The regulation of genes is far less well understood for morphological development than for biochemical differentiation. The responses of the four morphological traits described in this paper seem to offer a route to some greater insight into the genetic regulation of morphology.
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Hemifacial deficiency induced by a shift in dominance of the mouse mutation far: a possible genetic model for hemifacial microsomia.
Hemifacial deficiency appeared in 10% of juvenile mice when BALB/cGaBc mice carrying the recessive lethal mutation far were crossed with ICR/Bc. The hemifacial deficiency increased to 15-20% after one backcross to ICR/Bc and then remained at that level for 11 additional generations of backcrossing of far into ICR/Bc. Neither the ICR/Bc strain nor BALB/cGaBc (+/far) produces hemifacial deficiency. Genetic and anatomical studies of adults and fetuses showed that the hemifacial deficiency was due to +/far in the ICR/Bc strain genome; that is, far becomes an incomplete dominant in the ICR/Bc strain background. The hemifacial deficiency (38% of +/far) is probably caused by premature synostosis of the maxilla and premaxilla, observable on day 16 of gestation. An additional 20% of +/far in ICR/Bc have cleft palate and die at birth. Most +/far in both strains have a hidden anomaly, bilateral splitting of the maxillary branch of the trigeminal nerve. far/far homozygotes of both strain backgrounds have a syndrome of severe bilateral deficiency of the derivatives of the maxillary prominence. In human pedigrees, where the equivalents of the dominance modifiers in BALB/cGaBc and ICR/Bc would segregate within families, it would be difficult to recognize that sporadic hemifacial deficiency and severe bilateral maxillary deficiency were due to the same gene. We suggest that human bilateral and unilateral abnormalities of tissue derived from the first branchial arch should be analyzed with the awareness that, in mice, at least, the two kinds of syndrome are due to the same mutant gene.
Animals↗
Major genes that cause cleft lip in mice: progress in the construction of a congenic strain and in linkage mapping.
The major gene that causes cleft lip in the A/WySn mouse strain is being transferred to the unrelated normal AEJ/GnRk strain background by the backcross-testcross method. The result, a congenic strain pair differing only at the major cleft lip locus and closely linked loci, will allow the unequivocal identification of the genetic lesion that causes cleft lip. Four backcross generations have been completed, and the background geneotype is calculated to be 97% of AEJ/GnRk origin. The recovery of the cleft lip trait in testcrosses of each generation is compatible with the expected values for the transfer of a single major gene with 8% penetrance. Multilocus inheritance beyond two loci is ruled out. In separate experiments using mostly Robertsonian chromosomes as markers of the normal allele at the cleft lip locus, portions of the genome have been screened for the map location of the cleft lip gene. The gene is probably not within 10-20 cM of the centromere of 12 different identified autosomal chromosomes nor linked to Ph on chromosome 5. Previous studies have suggested that the cleft lip gene is not linked to c (chromosome 7), b (chromosome 4), d (chromosome 9), T, H-2 (chromosome 17), or N (chromosome 15).
Alleles↗
Eyelid development and fusion induced by cortisone treatment in mutant, lidgap-Miller, foetal mice. A scanning electron microscope study.
In normal mice the eyelids grow across the eye and fuse together during days 15 and 16 of gestation, and the mice are born with their eyes closed. In mutant lgMl)/lgMl) (lidgap-Miller) foetuses this growth and fusion does not occur and the mice are born with their eyes open. A single prenatal treatment with cortisone on day 14 of gestation masks the genetic defect, and the mice are born with their eyes closed. In the present study, a scanning electron microscope was used to investigate: whether eyelid closure in cortisone-treated lgMl/lgMl foetuses differs from that in normal foetuses of the CBA/J and ICR/Ml strains and, if so, how; and off developing eyelids of untreated lgMl/lgMl foetuses differ from normal. The induced closure differs from normal. During their growth, eyelids of treated mutant foetuses have a deficiency of rounded periderm cells at their margins; the margin cells agglomerate and flatten prematurely. In closed eyes of treated mutants there is a gap between periderm cells of the upper and lower eyelids along part of the fusion line. Fusion is completed late, during day 17. Maturation of the head periderm is advanced in treated lgMl/lgMl foetuses on day 16, relative to normal CBA/J and untreated lgMl/lgMl foetuses. Untreated lgMl/lgMl foetuses differ from normal in that the eyelids never cover the eye and lack rounded periderm cells at their perimeter. The periderm cells present form a flattened band along the eyelid margin rather than, as in normal eyelids, along the fusion line. The results of the cortisone study suggest that the route to eyelid closure in mice may not be narrowly canalized either in cell morphology or number, or in timing, and that closed eyes may be achieved after cortisone treatment in lgMl/lgMl foetuses as part of an induced maturation of the periderm.
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